1. Field of the Invention
The present invention relates to a regeneration medicine, more specifically a method of differentiation-inducing mesenchymal stem cells to osteoblasts.
2. Description of the Background Art
The regeneration medicine is a medicine aiming not only to regenerate cells, a tissue, and an organ of a living body lost by an accident or illness but also to recover the function of them. The medicine is one of the great challenges in the clinical medicine in the 21st century.
For example, after an external injury or a bone neoplasm is removed surgically, so far the bone tissue has been treated for the recovery by taking out an autologous bone, such as a thigh bone of the patient himself or herself, to transplant it to the affected portion. However, this method requires a surgical treatment for a sound thigh bone in addition to the treatment of the affected portion, giving the patient a doubled burden. Furthermore, the medical expense is increased.
On the other hand, the regeneration medicine, which has been attracting attention recently, is a medical technique that focuses attention on stem cells capable of differentiating to practically all tissues and organs in a living body. More specifically, by giving a specific instruction to stem cells from the outside, the stem cells are differentiation-induced to cells that construct a targeted tissue or organ. Thus, the medical technique finally regenerates the lost tissue or organ.
It is known that such stem cells exist in various types. They exist in nearly all organs and tissues in a living body. In particular, among various types of stem cells, such as haematopoietic stem cells and neural stem cells, cells known as embryonic stem (ES) cells can differentiate to any types of tissue and have high proliferating ability. Therefore, the EC cells are expected to be applied, as universal cells, to the treatment of various diseases such as Parkinson's disease, myocardial infarction, spinal damage, leukemia, diabetes, and liver disease.
However, the EC cells are cells produced from an early embryo (a fertilized egg) after an elapse of five to seven days or so from the time of fertilization in human and after an elapse of three to four days or so in mouse. Consequently, there is a problem of ethics. Therefore, there are many hurdles to cross before the cells are put into practical use in the regeneration medicine.
In place of the EC cells, another type of stem cells has been attracting attention recently, which is mesenchymal stem cells existing in the bone marrow. The mesenchymal stem cells have a potential to have an ability close to that of the EC cells. It has been confirmed that the mesenchymal stem cells turn into the cells of the bone, cartilage, fat, heart, nerve, liver, and so on. As a result, the mesenchymal stem cells attract attention as the second universal cells.
If the mesenchymal stem cells can be used, they can be differentiation-induced into the bone cells at the outside of a living body to treat a patient by transplanting the bone cells to the affected portion. Therefore, in the field of the regeneration medicine, the researchers have been studying by paying attention to how to take out mesenchymal stem cells with a reduced burden on the patient and how to proliferate the taken-out stem cells to such an extent that they can be transplanted.
For example, Patent literature 1 has disclosed that there exists cells, which differentiate to osteoblasts, in the amniotic epithelial cell layer of the human and that by separating the amnion from the placenta to culture the cells at the outside of the living body, the differentiation induction to osteoblasts can be achieved.
In addition, another Patent literature 2 has disclosed a method of separating fibroblasts in which, first, extracellular matrixes existing in a piece of fat in the human are digested by a collagenase treatment to obtain a group of cells, second, a group of mature adipocytes is separated by centrifugation, and, third, the performing of the primary culture differentiation-induces them to osteoblasts.
In addition to the above-described studies on the separation and culture of cells, researchers have been studying on the analysis of the differentiation mechanism of the mesenchymal stem cells. For example, Patent literature 3 has reported that the manifestation of a brain and muscle arnt-like protein 1 (BMAL1) gene, which is one of the biological clock-relevant factors, is recognized in the process of the differentiation to mast cells.                Patent literature 1: the published Japanese patent application Tokukai 2005-124460.        Patent literature 2: the published Japanese patent application Tokukai 2004-129549.        Patent literature 3: the published Japanese patent application Tokukai 2005-247740.        
However, the above-described Patent literature 1 has not stated that the cells that are supposed to be able to be differentiated to osteoblasts are mesenchymal stem cells. Instead, the literature has described that the cells are cells obtained by taking out from the amnion of the human. The literature has disclosed that because the human's amnion is a tissue derived from a fetal, the amnion can be easily taken out in a state that it is attached to the placenta of the mother's body. In addition, the literature has reported that because the fetal-derived amnion is used, the immune tolerance is manifested, so that even when it is transplanted to a different person, it has little tendency to cause rejection (see the paragraph [0013] in Patent literature 1). However, Class I of HLA is manifested. Therefore, it cannot be said that 100% immune tolerance is assured and consequently there is a risk that an unexpected rejection is caused after they are transplanted to the patient. Furthermore, the cells obtained from the amnion include cells that cannot be differentiation-induced to osteoblasts.
In addition, in the technique disclosed in Patent literature 2, the piece of fat can be taken out from the fat tissue of the patient himself or herself. Consequently, the rejection that Patent literature 1 may cause as a problem can be prevented. Nevertheless, as with Patent literature 1, Patent literature 2 has created a problem that cells that differentiate to osteoblasts cannot be separated with sufficient purity.
A problem common to Patent literatures 1 and 2 is that cells that differentiate to osteoblasts have not been able to be separated with sufficient purity. Consequently, even when the differentiation induction to osteoblasts has been successfully carried out at the outside of a living body, cells other than osteoblasts are also transplanted to the patient. Therefore, it is unclear whether or not the tissue or organ can be regenerated without problem as the one having a satisfactory function.
Furthermore, another problem common to Patent literatures 1 and 2 is that the separated cells require a few weeks for the preculture. In addition, to differentiation-induce the precultured cells to osteoblasts, the cells must be cultured at the outside of a living body for at least a few weeks by adding to the medium a specific differentiation-inducing factor, such as ascorbate, dexamethasone, and β-glycerol phosphoric acid.
In other words, the patient whose bone has been removed due to an external injury cannot be treated by the transplanting to the affected portion for at least one month. This is a considerably hard thing for the patient.
In both of Patent literatures 1 and 2, the switch for the differentiation induction is only the changing of the composition of the medium. If this approach is utilized in the future to a method in which only mesenchymal stem cells are transplanted into a living body to differentiate them later to osteoblasts, it will become necessary to administer a differentiation-inducing factor, such as ascorbate, dexamethasone, and β-glycerol phosphoric acid, to the affected portion that has been treated by the transplantation. Consequently, there exists a risk that the patient suffers an unexpected adverse reaction. Furthermore, as described above, mesenchymal stem cells have an ability to differentiate to the cells of the bone, cartilage, fat, heart, nerve, and liver. As a result, there is another risk that the foregoing differentiation-inducing factor exercises another function in the living body and differentiates to cells having another function at the affected portion.
In addition, Patent literature 3 has disclosed that the BMAL1 manifests itself in the process of differentiation to fat cells. However, the manifestation of the BMAL1 is controlled by using a technique in which a gene is introduced into the cells. Nevertheless, the technique for the control merely analyzes the mechanism of the manifestation of the BMAL1 at the molecular level. In other words, no novel findings have been disclosed on what type of instruction is to be given to mesenchymal stem cells in order to differentiate them to fat cells. The differentiation induction to fat cells are only performed by adding to the medium a differentiation-inducing factor, such as dexamethasone and isobutyl methylxanthine.